Open Access

Jordi Doijen, Koen Temmerman, Christel van der Eynde, Annick Diels, Nick van der Broeck, Michiel van Gool, Inha Heo, Steffen Jaensch, Marleen Zwaagstra, Mayra Diosa Toro, Winston Chiu, Steven De Jonghe, Pieter Leyssen, Denisa Bojkova, Sandra Ciesek, Jindrich Cinatl, Lore Verschueren, Christophe Buyck, Franciscus Johannes Maria van Kuppeveld, Johan Neyts, Marnix van Loock, Ellen van Damme

• The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.